Vandetanib is a novel multitarget tyrosine kinase inhibitor (TKI) that inhibits vascular endothelial growth factor receptor-2 (VEGFR-2), with additional inhibition of epidermal growth factor receptor (EGFR) and rearranged during transfection receptor signaling, which has shown promising results in clinical trials for advanced non-small cell lung cancer. However, the mechanisms of acquired resistance to vandetanib remain unclear. Therefore, we established in vitro vandetanib-resistant PC-9/VanR cells from PC-9, a vandetanib-sensitive lung adenocarcinoma cell line, by chronic exposure to this agent. PC-9/VanR cells were 50-fold more resistant to vandetanib than PC-9 cells in vitro. Compared with PC-9 cells, PC-9/VanR cells showed emergence of an EGFR T790M mutation, moderately elevated MET amplification, and similar VEGFR-2 inhibition by vandetanib. Note that phospho-MET in PC-9/VanR was suppressed following EGFR inhibition by an irreversible EGFR-TKI, indicating that MET signaling of PC-9/VanR was dependent on EGFR signaling and that MET amplification was not the primary mechanism of resistance to vandetanib. In contrast to the in vitro experiment, vandetanib effectively inhibited the growth of PC-9/VanR tumors in an in vivo xenograft model through the antiangiogenesis effects of VEGFR-2 inhibition. In conclusion, the multitarget TKI vandetanib induced or selected for the EGFR T790M mutation as observed previously with highly selective EGFR-TKIs. However, vandetanib retained significant efficacy in vivo against xenografts harboring the T790M mutation, providing a strong scientific rationale for investigating vandetanib in clinical settings where acquired resistance through emergence of EGFR T790M mutations limits the effectiveness of highly selective EGFR-TKIs.